Methionine is an essential amino acid in mammals that is required for protein synthesis. Methionine also plays a central role in metabolic reactions involving transfer of single-carbon moieties: in its activated form, S-adenosylmethionine, methionine is the methyl donor in hundreds of biological transmethylation reactions. Moreover, methionine is the propylamine donor in polyamine synthesis. The ultimate product resulting from the demethylation of methionine is homocysteine, the remethylation of which is catalyzed by a cobalamin-dependent enzyme, methionine synthase (5-methyltetrahydrofolate:homocysteine methyltransferase, EC 2.1.1.13).
The enzyme-bound cobalamin cofactor of methionine synthase plays an essential role in the methyl transfer reaction by acting as an intermediate methyl carrier between methyltetrahydrofolate and homocysteine. The upper portion of FIG. 1 illustrates the transfer of the methyl group of methyltetrahydrofolate (CH3—THF) to homocysteine via methionine synthase-methylcobalamin [MetSyn-CH3—Co(III)] as an intermediate methyl carrier. Cleavage of the methyl-cobalt bond of the methylcob(III)alamin intermediate occurs heterolytically so as to leave the cobalamin in the highly reactive cob(I)alamin oxidation state. The occasional oxidation of the enzyme-cobalamin to the cob(II)alamin state [MetSyn-Co(II)] renders the enzyme inactive.
Severe deficiency of methionine synthase activity leads to megaloblastic anemia, developmental delay, hyperhomocysteinemia, and hypomethioninemia. Moreover, elevated plasma homocysteine is a risk factor in cardiovascular disease and neural tube defects (Rozen, Clin. Invest. Med. 19:171–178, 1996).
Two forms of methionine synthase deficiency are known (Watkins et al., Am. J. Med. Genet. 34:427–434, 1989; Gulati et al., J. Biol. Chem. 272:19171–19175, 1997). The first is a primary defect of the amino acid sequence of the methionine synthase enzyme. We recently cloned cDNAs encoding human methionine synthase and showed that patients from the cblG complementation group of folate/cobalamin metabolism have mutations in the methionine synthase gene. A second class of patients, belonging to a distinct complementation group, cblE, is also deficient in methionine synthase enzymatic activity. The genetic basis of this deficiency has not been determined.
An analogous methylcobalamin-dependent methionine synthase has been well characterized in E. coli and the structures comprising its C-terminal half have been elucidated by X-ray crystallography. The reductive activation system required for its maintenance is a two-component flavoprotein system consisting of flavodoxin (a small FMN-containing electron transfer protein), and NADPH-ferredoxin (flavodoxin) oxidoreductase, a member of a family of electron transferases termed the “FNR family.” However, flavodoxins are not found in mammalian cells.
It would be desirable to identify the enzyme that catalyzes the reductive activation of methionine synthase, i.e., the methionine synthase reductase. Knowledge of the reductase wild-type nucleotide and amino acid sequences would allow the identification of mutations and polymorphisms associated with diseases involving methionine metabolism. Moreover, an understanding of the reductase structure and function will facilitate the identification of compounds that modulate its activity. Such compounds will be useful in treating and preventing disease and developmental defects.